The present invention relates to a method of detection, identification and/or quantification of bacteria and to test kits for carrying it out. Particularly the method enables detection of organisms of specific bacterial genus, species or serotype, in isolated form or as contaminants in environmental or forensic samples, or in foodstuffs.
There are many requirements for methods of screening for specific bacteria, particularly those present in low numbers and in specific environments; for example, human bacterial pathogens in contaminated foods. Public health and quality control bodies demand rapid bacterial detection methods which have suitable levels of specificity and sensitivity, but few satisfactory methods exist.
It is known to detect specific bacteria by use of genetically engineered bioluminescent bacteriophages, virus particles which have had the xe2x80x98luxxe2x80x99 gene inserted into their genome, as described by Ulitzer and Kuhn, in Scholuerich et al (Eds.) xe2x80x98Bioluminescence and Chemiluminesconce-new perspectivesxe2x80x99 pages 463-472; pub. (1987) by John Wiley and Sons. The xe2x80x98luxxe2x80x99 gene is that encoding for bacterial luciferase and the technique is based upon the fact that upon infection of a target bacterium, bacteriophage genes and the lux gene are injected into it and are subsequently expressed. The presence of the target bacterium is indicated by emission of light from the sample which can easily be measured. Most bacteria are susceptible to attack by bacteriophages (commonly called phages), many of which lyse or disrupt their host at the end of their replication process, and these interactions show varying degrees of host/phage specificity.
Schutzbank et al, The Use of xe2x80x9cBioluminescent Bacteriophagesxe2x80x9d for the Detection and Identification of Salmonella in Foods, Rapid Methods and Automation in Microfiology and Immunology, 1987, 5th ed., p 241-251, Berscia, Brixia Academic Press, 1987 have shown the potential or this technique but note problems with cross reactivity between construct phages and other non-target bacterial types. While such problems may be overcome by engineering more specific phages, see for example U.S. Pat. No. 4,348,478. this entails provision of phages for each type of target bacteria for which a need to test exists. Such recombinants may not readily be constructed for a variety of reasons, not least of which being the need to avoid disruption of the function of the phage itself.
Initially only Salmonella and E. coli phages were successfully modified. This is because a great deal of work had been previously done on the molecular biology of these organisms and it was known that the lux genes could fit into the phage head without causing lose of the phage""s ability to be infective. This is not the case with other food pathogens and a massive amount of work will need to be done before a comprehensive range of modified phages can be constructed.
A further method utilising the specificity of phages for their host bacteria is disclosed by Hirsh and Martin (1984) Journal of Food Protection, Vol 47, No 5, pp388-390. This method relies on the phage induced destruction of the bacteria grown on enrichment broth to produce lacunae in the bacterial lawns, wherein lacunae are indicative of the presence of the target bacterium; in this case a Salmonella. However the method is lengthy to perform, requiring about 24 hours for a positive result, and in only sensitive to 5 cells/ml or more.
The present inventor has now provided a method based upon the occurrence of release of cell contents on damage, e.g. lysis, of cell walls of bacteria when new phage particles are released at the end of the phage replication cycle. This method provides for the detection of specific bacteria which does not require insertion of the lux gene into the phage genome, whereby specificity is only limited by the availability of phage types suitable for selective attack of the target bacteria to be detected, yet its performance takes far less time than the existing unmodified phage techniques. Thus the method of the present invention has more readily realised potential for the specific and rapid detection of almost any bacteria in any environment; (foodstuffs, drinking water, pharmaceutical products and diseased tissues in humans, animals and plants etc.) provided that a phage with the necessary specificity can be found. Use of the method of the present invention has been shown to be capable of detection or a single Salmonella in a 1 ml sample of milk in under 12 hours.
Thus the present invention provides a method for the detection, identification and/or quantification of target organisms of specific bacterial genus, species or serotype present in a material comprising incubating the material or bacteria derived therefrom with bacteriophage selected for the ability to specifically infect target organisms and release cellular components from them, measuring the amount of one or more particular components released from any bacteria present during the incubation, and relating this to the presence, identity and/or amount of target organisms. Preferably incubation of material with bacteriophage is carried out in bacterial support medium.
Preferred cellular component comprises nucleotides. Theoretically it is possible to measure any or the nucleotides that are released by the cell lysis caused by the release of new phage particles, for example NAD, NADP, NADH, NADPH, ATP or ADP, cAMP or cGMP, with sensitivity provided by use of one or more of the many enzyme based assay systems, e.g. xe2x80x98cascadexe2x80x99 systems, that are available in the art. For example, GB 2213261 discloses a method which may be used for assaying reduced pyridine nucleotides, e.g. NADH or NADPH, based upon a salicylate monooxygenase system, while other enzyme systems such as the alkaline phosphatase/NAD/NADP system as disclosed in GB 2240845. Suitable assays for ADP, cAMP, cGMP etc will occur to those skilled in the art.
However, particularly preferred is the measurement of adenosine triphosphate (ATP), that being readily measurable by assay with a variety of enzyme/enzyme substrate combinations by virtue of its being a cofactor in numerous substrate conversions, and being released in relatively large quantities as compared with other bacterial nucleotides. For the rapid and efficient determination of levels of released ATP it is especially preferred to utilise enzyme reactions which result in production or luminescence, most conveniently using luciferase. ATP release is quantifiable with commercially available reagents using bioluminescence wherein it is used to drive oxidation of luciferin under catalysis by luciferase resulting in the emission of light. The quantum efficiency of this reaction is extremely high and the presence and amount of light produced gives a measure of ATP released and thus of the presence and numbers of target organisms.
For identification or quantification of specific bacteria occuring in material in relatively high concentrations, e.g. in cultures of isolated bacteria, it is possible merely to incubate the specific phage with a part of that culture or a subculture in the presence of, or with subsequent addition of, the component assay reagents and thus to measure the amount of component released by performing the assay.
For identification, detection and/or quantification of specific bacteria at lower concentrations, for example as contaminants in or on water or foodstuff materials, it is necessary to first perform an enrichment or bacteria derived from the material under test, e.g. for a few hours, to allow the target bacterium to multiply to a level where its released components, e.g. nucleotides, will be detectable above background levels. Enrichment is preferably carried out by culturing a selective medium, favouring growth of target bacteria, that has been inoculated with the material or a culture derived therefrom, e.g. derived from a swab. Typically enrichment lasts less than a working day, e.g. 1 to 10 hours; preferably 1 to 5 hours.
After enrichment, one or more phage types with known target host specificities are mixed with the culture and the mixture is incubated. During the incubation period infection or specific target bacteria with phage occurs and the replication cycle begins. At the end of the replication cycle target bacteria present burst (times range from e.g. 20-60 minutes depending on species), cellular component, e.g. nucleotide, is released and detected. Control samples either containing no bacteria, non-target bacteria or bacteria without phage show no substantial increase in levels above background levels.
Furthermore, dependent upon the biology of the phage/host pair selected, the method has the potential for great specificity and still further sensitivity and rapidity. Utilising the aforesaid ATP assay method the inventor has readily developed systems for the detection of several human pathogens in milk including Staphlococcus aureus, Listeria monocytogenes and Salmonella, and others for foodstuffs such as lettuce whereon pathogens such as Listeria are known to be a potential danger. Salmonella, Listeria, Staphylococci, E. coli and pseudomonads have all proved to be readily detectable by the method. It will be seen by those skilled in the art however that there is no limit to the application of the present method other that the availability of the necessary specific phage or phages.
In a more preferred embodiment of the present invention it has been found to be advantageous to incubate a sample of material under investigation with a nonselective bacterial support medium, e.g. such as peptone water or Brain Heart Infusion broth (B.H.I), in order to activate or xe2x80x98resuscitatexe2x80x99 substantially all or the bacteria present therein, before carrying out any enrichment incubation. Such incubation is conveniently, but not essentially, carried out for 1-5 hours; but as with the enrichment, shorter or longer periods may be appropriate depending on, inter alia, the freshness or the material.
In a preferred embodiment a sample of the resuscitated material is then transferred to a selective medium adapted to favour growth of the bacteria being investigated over interfering bacteria and incubated for a further period, e.g. 1-5 hours. Conveniently an aliquot of the medium from the resuscitation stage is so transferred.
After the incubations are complete all or part of the cultured media is mixed with the selected phage, e.g. phage in the form of a suspension or freeze dried, and incubated for a set time and at a set temperature selected according to the biological characteristics of the phage/bacteria combination being used. The amount of component, e.g. nucleotide, whose release is being measured is then measured using an appropriate assay, e.g. enzyme/substrate system.
For assaying the release of cellular component completed resuscitation or selective enrichment step medium, or a medium with a sample of the material itself, is conveniently mixed with the selected phage in a vessel, e.g. for ATP In a luminometer tube, and incubated for a suitable time and suitable temperature. e.g. 30 to 60 minutes at 20xc2x0 C. to 40xc2x0 C. to allow call lysis; these conditions depending on the phage/bacteria system. For measuring ATP, light producing assay reagents, e.g. those of the luciferin/luciferase system, can be added before or after this incubation period or the lysis period; light produced over the period or on addition is detected in a luminometer and related to the amount of ATP released. In all cases controls are advantageously carried out for comparison of background component levels, e.g. nucleotide levels, in samples incubated without the phage or incubated with phage and a known amount of target bacteria whereby production of calibration curves for assessing bacteria numbers is enabled. Such controls may include challenge with other phages of different specificity in order to more completely determine the characteristics of the organisms present in the material. Similarly several phage types may be used in the same incubation where the method is being used to screen for a number or types of bacteria for which no one common phage is specific.
The range of bacteriophages available and the bacteria for which they are specific will be realised to be vast by those skilled in the art. For example a list of phage types available from the American Type Culture Collection (ATCC) is published by them as xe2x80x98Catalogue of Bacteria and Bacteriophagesxe2x80x99. Other such depositories also publish equivalent data in their catalogues and this may be used to identify possible phage xe2x80x98reagentsxe2x80x99 for the present method. Phages may be used, inter alia, in aqueous suspension or in freezed dried form e.g. on microtitre plate wells. In this manner plate luminometry can be used. Preferred phages are lytic and lead to lysis of the target organism.
In addition to deposited phages, a further source or phages is provided by isolating them from suitable environments; that is conveniently the environment where the target bacteria are themselves to be found. For example, it is possible to isolate phages specific to both Campylobacter spp. and Salmonella spp. from effluent from a poultry processing plant. Isolation techniques will be well known to those skilled in the art but are exemplified by Loessner and Busse (1990) Applied and Environmental Microbiology Vol 56, pp1912-1918, and Adams xe2x80x98Bacteriophagesxe2x80x99 Pub Interaciance Inc (1959) pp447-455.
The range of media available for selective promotion or growth of a particular bacterial type will also be known to those skilled in the art and thee way function by positive action or by e.g. inhibition of other organisms. Examples of such media are illustrated by reference to supplier""s manuals, e.g. such as those available from UNIPATH Limited, Wade Road, basingstoke, HANTS, RG24 OPW, UK xe2x80x98Selective Microbiology for Food and Dairy Laboratoriesxe2x80x99, or e.g. the OXOID manual. These publications list, for example, media capable of favouring growth of Campylobacter, Listeria and Yersinia. Similarly methods for isolation or food pathogens for preparation or test samples are well known. (UNIPATH and OXOID are Registered Trade Marks).
The present invention also provides test kits for carrying out the method or the present invention and these are characterized in so far as they comprise a bacteriophage selected for the ability to specifically infect bacterial target organisms, ie. a type or types for which detection, identification and/or quantification is desired, in combination with some or all or the reagents which are specifically associated with the aforesaid method of the invention.
Thus preferably test kits of the present invention comprise; (a) a bacteriophage selected for its ability to specifically infect target bacterial organisms and release cellular components from them, preferably in a form substantially free of such target organisms, and (b) the reagents necessary for carrying out assay of the amount of one or more cellular components released by the action of the bacteriophage on the bacterial organism.
Optionally the kits further comprise (c) a selective medium for promoting preferential growth of target bacterial organisms and/or (d) a non-selective bacterial growth medium.
Thus preferred test kits of the invention are those wherein the reagents necessary for carrying out assay are for assay of the amount or a nucleotide present in a sample, preferably comprising luciferin and luciferase. Test kits optimised for performance of high sensitivity assay of bacteria will include both non-selective and selective growth media, the non-selective growth medium being preferably that suitable for a microorganism resuscitation step.
It will be realised that, unlike the prior art method which monitors lacunae on solid media supported bacterial lawns, the present method is particularly adapted to test for bacteria derived from samples of material under investigation by culturing on liquid media, although solid media may of course be used. A particular kit for performance of the present method may be one having the bacteriophage component (a) together with one or more media (c) and/or (d) of liquid form or concentrated or dry components adapted for the preparation of such liquid medium. While many such combinations adapted for the performance of the method of the present invention will occur to those skilled in the art, the most complete form of the kit will comprise all the reagents (a) to (d); the nucleotide assay components preferably being optimised for measuring nucleotide levels in a sample of the bacterial growth medium. Where quantification is desired samples or the bacteria might also be included, e.g. as spores or in cells in a medium, for setting up control readings of known levels of bacteria for calibrating the nucleotide assay. Target and control non-target organisms may be used.
The method and kits of the present invention will now be exemplified by way of illustration only by reference to the following non-limiting examples. The vast variety of options available will be readily determinable by those skilled in the art on consideration of the general method described above and particularised below, and the available types of bacteriophages and nucleotide assays.